1. Field of the Invention.
The present invention relates to an apparatus for the growth and maintenance of microorganisms or cells in culture.
2. Description of the Prior Art.
In order to grow microorganisms or cells in vitro, the environment in which the cells or microorganisms are maintained must be very carefully controlled. Variables, such as nutrients, gasses, and temperature must be maintained at the proper level for the cells or microorganisms to grow in an optimal manner. A nutrient solution containing CO.sub.2 and/or O.sub.2 is pumped into a culturing chamber in which the cells or microorganisms are held. The CO.sub.2 and/or O.sub.2 is used to control the pH of the nutrient solution and permit the cell or microorganism to properly respirate.
One prior art method of introducing CO.sub.2 and/or O.sub.2 into the nutrient solution is to gas the nutrient solution in its source container. Either the head-space in the source container is gassed or the nutrient solution is bubbled. Bubbling of the nutrient solution, however, tends to cause proteins to combine into larger aggregations which may change the fluid and biochemical properties of the nutrient solution. The nutrient solution is then delivered through silicone tubing to the culturing chamber. A pump is typically positioned between the nutrient source and the culturing chamber to provide the motive force to deliver the nutrient solution to the chamber. The silicone tubing is gas-diffusable and is well known, and since the distance from the source container to the culture chamber is relatively long and the flow rate relatively slow, much of the gas within the nutrient solution diffuses out of the silicone tubing.
Another method of introducing CO.sub.2 and/or O.sub.2 into the nutrient solution is to introduce gas directly into the culture chamber. The culture chamber is only partially filled with the nutrient solution, creating a head-space within the chamber. The head-space is then directly filled with gas. Vessel geometry is consequently critical because of the need to maintain an optimum surface area to total liquid volume ratio. In addition, agitation is also critical since the surface liquid must be circulated downward while the liquid at the bottom of the chamber must be brought to the surface (or at least into contact with highly gassed surface liquid) in order for the nutrient solution to be gassed properly. However, adverse conditions such as overgassing and cell damage due to surface tension and osmotic gradients occur at or proximate the air/liquid interface and mechanical agitation of the culture chamber may produce cell damage.
Microorganisms and cells are maintained at a predetermined temperature for growth. The culturing chamber is commonly kept in a water bath or air incubator which is held at a constant temperature. To avoid thermally shocking the microorganisms or cells with a nutrient solution having a temperature different than the culturing chamber, the incoming nutrient solution is typically prewarmed by passing it through a length of tubing (silicone) which is submerged in the same water bath as the chamber. This method, however, is difficult to use when sterility must be maintained or a sterile culture recovery is required. Furthermore, the silicone tubing acts as a membrane through which some ion transfer may occur. The ion transfer results in uncontrolled modification of the chemistry of the nutrient solution.
Another method that has been used to heat and gas the nutrient solution includes using a gassed incubator that may include a static culture, or a batch exchange culture, or a perifusion system with pumps, tubing and culture chambers within the incubator. This method, although eliminating many of the problems of the water bath described above, requires a relatively large amount of space, especially when using a perifusion system.
Other attempts have been made to heat the nutrient solution through the use of electric heaters. However, a significant amount of heat loss occurs through the tubing and creates a problem in trying to keep the temperature of the incoming nutrient solution the same as the temperature in the culture chamber. In addition, sophisticated controller systems that attempt to monitor both the culture chamber and the warmed nutrient solution and control the temperature of the incoming nutrient solution are costly, complex, require constant calibration, and are generally ill suited to the small volumes and low flow rates used in most tissue culture systems.